This invention relates to Streptococcus agalactiae, Group B Streptococcus, and in particular this invention relates to the identification of a S. agalactiae polypeptide and a gene encoding it that may be implicated in Group B streptococcal adhesion and/or virulence. The S. agalactiae polypeptide according to the invention demonstrates binding of human complement C3.
Streptococcus agalactiae, or group B Streptococcus (GBS), is a leading cause of infant mortality. GBS encompasses an estimated prevalence of several thousand cases per year resulting in an annual mortality rate in the United States between about 10% and 15% (Schuchat, Clin. Micro Rev., 11(3):497-513 (1998)). Although worldwide prevalence is known, insufficient specific epidemiological data is not readily available.
Several virulence factors have been reported in GBS,. In addition to the streptococcal capsule, which is an important virulence factor, lipotechoic acid, a glycerol-phosphate polymer extending throughout the cell wall, is a virulence factor that may mediate adhesion (Teti et al., Infection and Immunity, 55(12):3057-3064 (1987)). Proteins such as hyaluronate lyase, cAMP factor, proteases, nucleases, hippuricase, neuraminidase, hemolysin, and C5a peptidase are expressed from GBS and many have been shown to be virulence factors (Nizet et al., Streptococcal Infections, Stevens D L and Kaplan E L, Eds. (2000); Bohnsack et al., Biochimica et Biophysica Acta, 1079:222-228 (1991)). In short, many studies are underway to define virulence factors molecularly by the mutation of genes encoding these proteins under question and the examination in assays for biological function.
The C proteins, or antigens, have been further characterized as alpha proteins (Madoff et al., Infection and Immunity, 59(1):204-210 (1991)), as beta proteins (Russell-Jones et al., J. Exp Med. 160:1467-1475 (1984); Jerlstrom et al., Mol. Microbiol., 5:843-849 (1991)), and as gamma and delta proteins (Brady et al., Infect. Immun., 57:1573-1581 (1989)). Not much is known about gamma and delta C proteins as there have not been further reports since their identification. The alpha C protein, which is resistant to the protease trypsin, varies in molecular weight from about 30 kDa to about 190 kDa in a ladder-like array (Madoff et al., Infection and Immunity, 59(1):204-210 (1991)). The beta C protein, which is sensitive to the protease trypsin and typically exhibits less variability in size (molecular weight of about 14 kDa to about 145 kDa), is typically expressed as a single 130 kDa protein that is capable of binding human IgA (Russell-Jones et al., J. Exp. Med., 160:1467-1475 (1984)).
C proteins are suggested to contribute to GBS virulence in infant rat models (Ferrieri et al., Infection and Immunity, 27(3):1023-1032 (1980)) and some GBS strains without C protein are more easily killed in an opsonophagocytic bactericidal assay (Payne and Ferrieri, J Infectious Diseases, 151(4):672-681 (1985)). Other alpha-like proteins have also been reported (Kvam et al, Pathogenic Streptococci: Present and Future, Lancer Publication (A. Totolian (Ed.), St. Petersburg, Russia (1994)), and are under further investigation.
R proteins, or antigens, first identified by Lancefield and Perlman, were further characterized (Wilkinson, Infection and Immunity, 4(5):596-604 (1971); Bevanger et al., APMIS, 103:731-736 (1995)) and later determined to resemble the alpha C proteins in multiple molecular weight forms, resistance to trypsin, and imunogenicity (Wilkinson, Infection and Immunity, 4(5):596-604 (1971); Madoff et al., Infection and Immunity, 59(8):2638-2644 (1991); Flores and Ferrieri, Zbl.Bakt., 285:44-51 (1996)). The R proteins were identified as separate proteins R1, R2, R3, R4, and R5 by various investigators (Flores and Ferrieri, Zbl.Bakt., 285:44-51 (1996); Wilkinson, Applied Microbiology, 24(4):669-670 (1972)). Finally, a similar protein designated xe2x80x9cRibxe2x80x9d (Stalhammar-Carlemalm et al., J. Experimental Medicine, 177:1593-1603 (1993)), while proposed as an individual entity, is suspected to be an R protein (Flores and Ferrieri, Zbl.Bakt., 285:44-51 (1996)). Whereas these proteins have been characterized and grouped phenotypically, further molecular characterization of these groups is underway as well as the identification of new surface proteins unique in structure and biological functions.
Human mothers colonized with GBS represent approximately 15-35% of the U.S. population, and are known to transmit GBS to their infants before birth. This results in an incidence of neonatal GBS disease at a rate of 1 to 2 infants per 1,000 live births, and a mortality after birth due to complications of bacteremia/sepsis, and/or meningitis, or GBS infection in utero possibly resulting in stillbirth (Schuchat, Clin. Micro Rev., 11(3):497-513 (1998)). Even infant survivors of GBS meningitis suffer from resulting chronic neurologic injury ranging from deafness, learning disabilities, as well as motor, sensory, and cognitive impairment (Baker et al., Infectious Diseases of the Fetus and Newborn Infant., (4th Ed.) W.B. Saunders Company (1995)). Currently, antibiotic prophylaxis in parturients is the recommended approach for prevention of neonatal disease; however, this approach may be ineffective. With the resurgence of antibiotic resistance in other streptococcal species, a similar plight in group B Streptococcus may occur, making the need for effective vaccines urgent. It is known that infants do not make sufficient protective antibodies to GBS capsular polysaccharides, and maternal antibodies to capsule may not be sufficient for placental transfer and protection. Even after vaccination with current polysaccharide vaccines, antibody titers are low in individuals at greatest risk for colonization and severe infection (Linden et al., Int. Archs. Allergy Appl. Immun., 71:168-172 (1983)).
In addition to infants, other persons at high risk for GBS infection are the elderly and immuno-compromised persons (Farley et al., N. Engl. J. Med., 328:1807-1811 (1993)). Although currently under investigation, sufficient data on antibody production in the elderly is lacking. Antibody titers, however, may also be deficient in this age group, and it remains possible that immunization may protect elderly against invasive GBS disease (Schuchat, Clin. Micro Rev., 11(3):497-513 (1998)).
In addition to the problems confronting humans, many GBS strains are known to cause bovine mastitis in cows resulting in a monetary loss to dairy farmers as well as GBS strains known to cause mastitis in goats and other lactating mammals. GBS in cows is currently controlled by prophylactic antibiotic treatment (Keefe, Can. Vet. J., 38(7):429-37 (1997)). Concerns, however, have been raised regarding residual antimicrobials in milk from this prophylactic treatment, as well as the growing antimicrobial resistance in GBS strains (Baynes et al., J Food Prot., 62(2):177-80(1999)).
In response to the dilemmas described above, protein conjugate vaccines have been developed, and several are currently in clinical trials (Larsson et al., Infection and Immunity, 64(9):3518-3523 (1996)). Proteins that could be used as components of vaccines, as conjugates coupled to capsular polysaccharide (which act as adjuvants to boost antibody response), or as whole protein vaccine candidates themselves are under investigation. Problems existing with some protein conjugate vaccines include variability in protein structure resulting in a ladder-like array of proteins (Gravekamp et al., Infection and Immunity., 65(12):5216-5221 (1997)).
Another significant issue is that current protein conjugate vaccines are restricted to the GBS serotypes prevalent only in the United States. This includes mainly types I, II, III, and V, in contrast to a range of nine different serotypes that are able to colonize and cause invasive disease (Harrison et al., J. of Infectious Disease, 177:998-1002 (1998)). Since 1990, four new capsular serotypes IV, V, VI, and VII have been identified as associated with human invasive disease, with an increasing prevalence in type V (Blumberg et al., J. of Infectious Disease 173:365-73 (1996)). In addition, types VI and VIII are the most common serotypes in Japan not protected against by current vaccines (Lachenauer et al., J Infectious Disease, 179:1030-1033 (1999)).
Thus, a need exists for polypeptides that can be employed in immunogenic compositions and vaccines, for example.
A novel group B streptococcal protein GBbcA, Group B binds complement C3 has been identified. GBbcA is a surface polypeptide found in all group B streptococcalstrains and serotypes examined to date that bind complement protein C3. In addition to its surface expression and conservation, it appears to be immunogenic in both rabbits and humans, thus fulfilling certain criteria for vaccine candidacy. In addition, data suggests that antibodies are placentally transferred from mother to neonate, which is especially important in the development of vaccines and immunogenic compositions for pediactric diseases.
The present invention relates to the identification and isolation of a 60 kDaxc2x110 kDa polypeptide as determined by electrophoresis on a 7.5% SDS-PAGE gel. A preferred polypeptide is named GBbcA, and can be isolated from S. agalactiae strains that bind to human complement protein C3. The polypeptide GBbcA (SEQ ID NO:5, FIG. 10) has an internal peptide region (SEQ ID NO:2, FIG. 11).
Accordingly, the present invention provides an isolated polypeptide having at least 50% amino acid identity with the polypeptide represented by SEQ ID NO:5. The isolated polypeptide of the invention can be longer, shorter, or of the same length of amino acids as the polypeptide represented by SEQ ID NO:5. Preferably, the polypeptide binds human complement C3 and is isolated from S. agalactiae. Additionally, the polypeptide of the invention can be a recombinant polypeptide. The polypeptide preferably has a molecular weight of about 50 kDa to about 70 kDa as determined by SDS-polyacrylamide gel electrophoresis.
The present invention further provides a polypeptide that binds human complement C3 containing amino acids 365-370 of SEQ ID NO:5. Further, an isolated polypeptide may include amino acids represented by SEQ ID NO:1 wherein the polypeptide binds human complement C3.
In another aspect of the invention, an isolated polypeptide, which has a molecular weight of about 50 kDa to about 70 kDa as determined by SDS-polyacrylamide gel electrophoresis; is isolated from S. agalactiae, and binds human complement C3, is provided. A preferred polypeptide having these characteristics is represented by SEQ ID NO:5. Further, an isolated polypeptide containing the amino acids represented by SEQ ID NO:5 wherein the polypeptide binds human complement C3 is also provided.
In yet another aspect of the invention, an isolated polypeptide that binds human complement C3, wherein nucleic acid encoding the polypeptide hybridizes to at least a portion of at least one of the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands under standard hybridization conditions, is provided. The nucleic acid fragments can be longer, shorter, or of the same length of nucleotides as the polynucleotides represented by SEQ ID NO:6 or SEQ ID NO:4. Preferably, the nucleic acid encoding the polypeptide has at least 50% nucleic acid identity to the nucleic acid fragments represented by SEQ ID NO:4 or SEQ ID NO:6.
In another aspect of the invention, an isolated polypeptide of about 50 kDa to about 70 kDa from S. agalactiae that binds human complement C3 is provided.
The present invention further provides an immune system stimulating composition containing an effective amount of an immune system stimulating polypeptide, wherein the polypeptide has at least 50% amino acid identity with the polypeptide represented by SEQ ID NO:5 and binds human complement C3. Preferably, the polypeptide of the immune system stimulating composition is isolated from S. agalactiae. Additionally, the immune system stimulating composition can contain at least one other immune system stimulating polypeptide isolated from S. agalactiae. 
In another aspect of the invention, an immune system stimulating composition containing an effective amount of a polypeptide, wherein nucleic acid encoding the polypeptide hybridizes to at least a portion of at least one of the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands under standard hybridization conditions is provided. Preferably, an immune system stimulating composition according to the invention contains an effective amount of at least a portion of the about 50 kDa to about 70 kDa polypeptide that is effective to treat a mammal against S. agalactiae colonization or infection, and a pharmaceutically acceptable carrier.
In yet another aspect of the invention, an antibody that binds to a polypeptide which binds human complement C3 having at least 50% amino acid identity with the polypeptide represented by SEQ ID NO:5 is provided. The antibody can be obtained from a mouse, a rat, a goat, a chicken, a human, or a rabbit. Preferably, the antibody is a monoclonal antibody.
The present invention further provides an antibody that binds a polypeptide, wherein nucleic acid encoding the polypeptide hybridizes to at least a portion of at least one the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands under standard hybridization conditions.
In another aspect of the invention, an isolated nucleic acid fragment that hybridizes to at least a portion of at least one the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands under standard hybridization conditions is provided. The nucleic acid fragment can be isolated from S. agalactiae and preferably encodes a polypeptide represented by SEQ ID NO:5. Preferably, the polypeptide binds human complement C3. The nucleic acid fragment of the invention can further be provided in a nucleic acid vector, such as an expression vector and is capable of producing a polypeptide as described herein. Nucleic acid fragments of the invention can be provided in a cell, such as a bacterium or a eukaryotic cell.
In another aspect of the invention, an isolated nucleic acid having at least 50% nucleic acid identity to the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 and which hybridizes under standard hybridization conditions to at least a portion of at least one of the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands is provided.
Also provided is an isolated polynucleotide encoding a polypeptide containing the amino acids represented by SEQ ID NO:5. Further provided is an isolated nucleic acid fragment containing the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands. Additionally, an RNA transcribed by a double-stranded nucleic acid containing nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands is provided.
In yet another aspect of the invention, a method for producing an immune response to S. agalactiae in a mammal having the steps of administering a composition containing an effective amount of a polypeptide to a mammal, wherein nucleic acid encoding the polypeptide hybridizes to at least a portion of at least one of the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands under standard hybridization conditions in a pharmaceutically acceptable carrier to yield an immune response is provided. The immune response can be a B cell response, a T cell response, an epithelial cell response, or an endothelial cell response. Additionally, the composition may further contain at least one other immune system stimulating polypeptide from S. agalactiae. 
The present invention also provides a method for producing an immune response to S. agalactiae in a mammal having the steps of administering a composition containing an effective amount of a polypeptide to a mammal, wherein nucleic acid encoding the polypeptide hybridizes to at least a portion of at least one of the nucleic acid fragments represented by SEQ ID NO:6 or SEQ ID NO:4 or their complementary strands under standard hybridization conditions in a pharmaceutically acceptable carrier to yield an immune response.
In yet another aspect of the invention, an antigenic conjugate molecule having a capsular polysaccharide derived from Group B Streptococcus and a polypeptide containing the amino acids represented by SEQ ID NO:5 wherein two or more side chains terminating in sialic acid residues of the polysaccharide component are each linked through a secondary amine bond to polypeptide is provided. Also provided is a method of immunizing a mammal wherein a vaccine containing the antigenic conjugate molecule is administered in an effect amount to a mammal at risk for being colonized or infected by Group B Streptococcus.
The present invention also provides a method for detecting Group B Streptococcus in a mammal containing, (a) contacting an amount of an isolated nucleic acid from a biological sample from a mammal exposed to or afflicted with Group B Streptococcus colonization or infection with an amount of at least two oligonucleotides under conditions effective to amplify the nucleic acid so as to yield an amount of an amplified nucleic acid, wherein at least one oligonucleotide is specific for the nucleic acid represented by either SEQ ID NO:4 or SEQ ID NO:6 or their complementary strands; and (b) detecting the presence of the amplified nucleic acid, wherein the presence of the amplified nucleic acid is indicative of a mammal exposed to or afflicted with Group B Streptococcus colonization or infection.
As used herein the terms xe2x80x9cisolated and/or isolatable,xe2x80x9d when referring to a nucleic acid fragment means that it has been removed from a sample in which it is originally found. This may include concentrating the desired nucleic acid fragment without necessarily removing any other materials. It also includes separating nucleic acid fragments from cells expressing the nucleic acid fragments or from other materials, such as cellular components, polypeptides, lipids, salts, etc. In referring to a polypeptide that is xe2x80x9cisolated and/or isolatable,xe2x80x9d either removed from its natural environment or synthetically derived. Preferably, it is meant that the polypeptide is purified, i.e., essentially free from any other polypeptides and associated cellular products or other impurities.
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide; protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
xe2x80x9cNucleic acid fragmentxe2x80x9d or xe2x80x9cpolynucleotidexe2x80x9d as used herein refers to a linear polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A nucleic acid fragment may include both coding and noncoding regions that can be obtained directly from a natural source (e.g., a microorganism), or can be prepared with the aid of recombinant or synthetic techniques. A nucleic acid according to the invention may be equivalent to this nucleic acid fragment or it can include this fragment in addition to, one or more other nucleotides. For example, the nucleic acid fragment of the invention can be a vector, such as an expression or cloning vector.
xe2x80x9cPercentage amino acid identityxe2x80x9d refers to a comparison of the amino acids of two polypeptides as described herein. Amino acid alignment may be determined, for example, using the sequence alignment program CLUSTAL W available on the Internet at genome.ad.jp/SIT/CLUSTALW.html and percent amino acid identity may be determined by BLAST at National Center for Biotechnology Information (NCBI) website available on the Internet at ncbi.nlm.nih.gov.
xe2x80x9cPercentage nucleic acid identityxe2x80x9d refers to a comparison of the nucleic acids of two nucleic acid fragments as described herein. Nucleic acid alignment may be determined, for example, using a nucleic acid alignment program available on the Internet at genome.ad.jp/SIT/CLUSTALW.html and percent nucleic acid identity may be determined by BLAST at National Center for Biotechnology Information (NCBI) website available on the Internet at ncbi.nlm.nih.gov.
xe2x80x9cStandard hybridization conditionsxe2x80x9d refers to hybridizing conditions of prehybridization for 1 hour at 62xc2x0 C. in hybridization solution (5xc3x97SSC (1xc3x97SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.02% sodium dodecyl sulfate (SDS), 0.1% N-lauroylsarcosine, 1% Blocking Reagent) and subsequent hybridization with the 1337 basepair gbbcA digoxygenin-probe (1000 ng/ml) in the hybridization solution overnight at 62xc2x0 C. and two stringency washes with 2xc3x97SSC, 0.1% SDS for 5 minutes at room temperature and once with 0.5xc3x97SSC, 0.1% SDS for 15 minutes at 62xc2x0 C. followed by a digoxygenin detection protocol pursuant to manufacturer""s directions (Roche Molecular Biochemicals, Indianapolis, Ind.).